DETERMINATION OF DNA AND OF OTHER NUCLEIC ACID FRACTIONS

J.
Inst.
Brew.,
July-August,
1980,
Vol.
86,/y?.
171-173
171
DETERMINATION
OF
DNA
AND
OF
OTHER
NUCLEIC
ACID
FRACTIONS
IN
SACCHAROMYCES
CEREVISIAE
By
William
E.
Trevelyan
{Tropical
Products
Institute,
56-62
Gray's Inn
Road,

London
WC1X8LU)
Received
24
October
1979
After
extraction
overnight
of
Saccharomyces
cerevisiae
(baker's
yeast)
with
dilute
alkali,
to
remove
nucleotide
pools
and
RNA,
acidification
gave
an
insoluble
residue
from
which

DNA
guanine
and
adenine
were
extracted,
in
the
free
form,
by
1M
perchloric
acid
at
room
temperature,
and
deter
mined
by
cation-exchange
chromatography.
Percentage
DNA
in
the
yeast
was
computed

as
0062
x
pmol/g
of
guanine
+
adenine.
Preliminary
extraction
of
nucleotides
was
unnecessary,
and
gave
low
results
if
acid
reagents
(perchloric
or
trichloroacetic
acids)
were
used.
Several
reagents
were

tested
for
their
ability
to
extract
the
nucleotide
pool
from
intact
yeast
without
significant
effect
on
cell
RNA
content.
The
content
of
guanine
+
adenine
in
pool-free
yeast
was
taken as

giving
the
sum
of
RNA
+
DNA
purines,
RNA
purines
being
found
by
difference.
Key
words:
yeast,
analysis,
DNA,
nucleic
acids,
purines.
Introduction
The
nucleic
acids
of
yeast1-
have
technological

as
well
as
scientific
importance.
Single-cell
protein,
of
which
the
brewing
industry
is
one
of
the
largest
producers,13
has
to
be
processed
to
reduce
its
high
content
of
nucleic
acid

(RNA)
if
it
is
to
be
suitable
for
human
consumption,8
though
the
same
problem
does
not
arise
with
animal
feeds.14
Yeast
is
also
a
source
of
ribonucleic
acid
used
in

the
production
of
flavour
nucleo
tides.7
DNA,
which
occurs
in
yeast
in
relatively
small
amount,
is
not
produced
in
bulk;
but
its
manipulation,
by
the
tech
niques
of
genetic
engineering,

is
now
attracting
considerable
attention,20
while
the
amount
per
cell,
and
the
base
composi
tion
(%
GQ,
of
yeast
DNA
have
important
implications,
e.g.,
in
taxonomy."
The
DNA
content
of

yeast
is
usually
determined,
after
its
extraction
from
yeast
with
hot,
dilute
perchloric
acid,10
by
what
Carter1
describes
as
'.
. .
the
laborious
and somewhat
inaccurate
diphenylamine
method'.
An
alternative
method

was
suggested
by
the
rinding
that
treatment
of
yeast
with
Im
perchloric
acid
(PCA)
for
18
h
at
room
temperature
de-
purinated
its
DNA,
releasing
free
adenine
and
guaninc,
in

the
molar
ratio
of
1-7:1,
characteristic
of
yeast
deoxyribonucleic
acid.10
The
DNA
content,
as
estimated
from
the
amount
of
free
adenine
+
guanine
extracted
by
PCA,
agreed
with
that
given

by
the
standard
diphenylamine
procedure
in
the
case
of
yeast
preparations
from
which
nearly
all
the
RNA
had
been
removed
by
autolysis.
Unfortunately,
RNA
is
depurinated
to
some
extent
by

IM
PCA,
so
that
the
method
failed
when
it
was
applied
to
the
original,
non-autolysed
yeast,
which
contained
about
twenty
times
as
much
RNA
as
DNA.
It
has
now
been

found
that
RNA
can
be
efficiently
removed
from
the
cells
of
Saccharomyces
cerevisiae
by
dilute
sodium
hydroxide,
and
remains
in
solution
when
DNA
(largely,
but
not
entirely,
bound
to
the

cells)
is
precipitated
by
acid
(Schmidt-Thannhauser
procedure).
DNA
can
then
be
readily
determined
after
extraction
of
the
insoluble
residue
with
1m
perchloric
acid.
RNA,
the
major
nucleic
acid
fraction
of

yeast,
can
also
be
determined
by
purinc
analysis.
The
nucleotide
pool
is
first
re
moved
from
a
duplicate
sample
of
yeast
by
any
of
a
number
of
possible
methods,
when

1m
perchloric
acid
at
room
tempera
ture
extracts
from
the
pool-free
cells
both
the
purines
of
DNA,
and
partially
degraded
RNA
in
the
form
of
an
ill-defined
mixture
of
oligonucleotidcs.

Hydrolysis,
by
heating
the
extract
for
1
h
at
100°C,
liberates
all
purines
in
the
free
form,
allowing
the
determination
of
the
sum
of
the
purines
of
DNA
and
RNA.

The
content
of
RNA
purines,
and
hence
the
amount
of
RNA
in
the
yeast,
is
then
obtained
by
difference.
All
the
methods
for
extracting the
nucleotide
pool
which
were
tried
failed

to
effect
an
absolute
separation
of
nucleo
tides
from
nucleic
acids,
though
this
did
not
cause
any
serious
error
in
the
determination
of
RNA
content.
Methods
Purine
estimation.—A
2-5
ml

sample
loop,
made
by
connect
ing
two
3-way
valves
(Pharmacia
LV-3)
with
the
appropriate
length
of
1
-S
mm
bore
PTFE
tubing,
was
charged
with
the
sample.
One
end
of

the
loop
was
then
connected
to
a
peristaltic
pump,
which
delivered
40
ml/h
of
a
filtered,
de-aerated
solu
tion
of
K.H2POi(0-5M,pH
4-3),
and
the
other
to
a 110
x
10mm
column

of
Bio-Rad
AG
SOW
X4
cation-exchange
resin,
main
tained
at
32°C.
Pyrimidine
nuclcotides
were
eluted
early
on,
and
followed
by
guanine
and
then
adenine
in
well-defined
peaks,
which
were
recorded

by an
ISCO
Model
UA5
ultra
violet
monitor
(254
nm).
After
3
h,
the
column
was
ready
to
receive
the
next
sample.
Over
a
period
of
several
days,
peak
heights
were

reproducible
to
within
1
mm,
corresponding
to
about
0-1
ixmol
purinc/g
yeast
dry
matter
(DM)
in
the
case
of
DNA
analysis.
Comparison
was
made
with
the
peak
heights
given
by

a
standard
solution
of
guanine
+
adenine,17
0-5
/xmol/ml
in
Im
PCA,
which
was
mixed
with
an
equal
volume
of
Im
K2HPO4
and,
after
the
precipitate
of
potassium
per-
chlorate

had
been
removed,
diluted
appropriately
with
0-5m
KH2PO4.
Yeast.—Eighteen
g
of
commercial
pressed
baker's
yeast
(Distillers
Company
Ltd)
was
washed
three
times
in
succession
by
centrifuging
a
suspension
in
60

ml
water,
and
made
to
100
ml.
Five
ml
of
suspension,
delivered
by
pipetting
syringe,
contained
approximately
250
mg
of
dry matter
(residue
after
drying
for
20
h
at
IO5°C);
duplicate

determinations
differed
by
0-3
mg
(mean,
N
=
9).
Total
purine
content.—Twenty
ml
of
1-25m
PCA
were
added
to
S
ml
of
yeast
suspension
contained
in
a
screw-capped
(universal)
bottle;

this
was
clipped
to
a
motor-driven
shaft
and
rotated
end-over-end
for
18
h.
The
yeast
cells
were
sedimented
by
centrifuging,
and
10
ml
of
the
supernatant
heated
for
1
h

at
100°C
(water-bath,
screw-capped
tube)
to
obtain
purines
in
the
free
form.
Before
the solution
was
applied
to
the
column,
perchlorate
was
precipitated
with
K2HPO4,
as
above.
Neglect
of
the
volume

occupied
by
the
yeast
solids
intro
duced
an
error
of
less
than
1
%.
An
alternative
procedure,
convenient
when
the
yeast
sample
was
in
the
form
of
a
centrifuged
pellet,

was
to
extract
twice
with
Im
PCA
(10
ml
for
18
h,
then
10
ml
for
0-5
h;
combined
supernatants
+
2-5
ml
of
2m
PCA
diluted
to
25
ml).

Extraction
of
nucleotide
pool.—Several
methods
were
tried,
of
which
the
following
gave
reproducible
results.
(A).
Five
ml
of
yeast
suspension
were
delivered
into
a
thin-walled
tube,
and
stirred
by
means

of
a
2
mm
glass
rod
attached
by
a
flexible
coupling
to the
shaft
of
an
Electrothermal
Mini-
stirrer.
The
tube
was
immersed
for
5
min
in
a
bath
of
water

at
80°C,
and
then
cooled
in
ice.
The
stirrer
was
washed
off
with
5
ml
of
water; then
the
yeast
was
recovered
by
centrifuging,
and
re-extracted for
10
min
with 10
ml
of

cold
water.
(B).
The
sample
of
yeast
suspension
was
mixed
end-over-end
for
15
min
with
5
ml
of
trichloroacetatc
buffer
(1
mol
TCA
+
0-5
mol
NaOH/litrc),
centrifuged,
and
the

cells
re-extracted
172
trevelyan
:
determination
of
dna
in
Saccharomyces
cerevisiae
[J.
Inst.
Brew.
with
10
ml
of water
(10
min).
(C).
The
sample
was
extracted
for
IS
min
with
5

ml
of
a
solution
containing
0-4
mol
of
perchloric
acid
-(-
4-0
mol
of
acetic
acid/litre,
and
re-extracted
with
10
ml
of
0-2m
PCA.
(D).
The
sample
was
centrifuged
and

the
supernatant
discarded.
With
a
glass
rod,
the
yeast
pellet
was
stirred
for
5
min
with
1
ml
of
2m
PCA;
the
rod
was
washed
off
with
8
ml
of

water,
the
suspension
mixed
end-over-
end
for
IS
min,
the
yeast
centrifuged
down
and
re-extracted
with
10ml
of
02m
PCA.
To
determine
total
guanine
+
adenine
in
the
extracts,
they

were
made
1m
with
respect
to
PCA,
and
hydrolysis
carried
out
at
100°C,
as
above.
When
TCA
was
the
cxtractant,
this
had
to
be
done
in
an open
tube,
to
allow

the
escape
of
chloroform
resulting
from
the
decomposition
of
the
acid.
DNA
+
RNA
purine
content.—With
the
aid
of
a
plastic
Pasteur
pipette,
the
pellet
of
pool-free
yeast
cells
was

trans
ferred
(in
water)
to
a
25
ml
volumetric
flask.
Five
ml
of
5m
PCA
were
added,
followed
by
water
to the
mark.
The
suspen
sion
was
poured
into
a
screw-capped

bottle
and
then
treated
as
under
total
purine
content.
Alternatively,
the
successive
extrac
tion
procedure
was
used.
DNA
purine
content.—Five
ml
of
yeast
suspension
(or
250
mg
of
freeze-dried
yeast)

were
measured
into
a
IS
ml
polypropylene
centrifuge
tube;
2-5
ml
of
1m
NaOH
were
added,
and
water
to
10
ml.
The
tube
was
stoppered,
and
the
contents
agitated
gently

overnight
(end-over-end
mixer).
Perchloric
acid
(2-5
ml
of
a
1-5m
solution)
was
added,
and
the
suspension
mixed
for
10
min.
The
insoluble
residue
was
centrifuged
down,
washed
twice
with
10

ml
of
01m
PCA,
and
extracted
overnight
with
8
ml
of
1m
PCA.
The
supernatant
was
decanted
into
a
25
ml
graduated
cylinder,
and
followed
by
a
second
extract
(4

ml Im
PCA
-t-
4
ml
water,
30
min);
the
combined
extracts
were
made
to
17
ml.
Then
8
ml
of
1-5m
K2HPO4
were
added,
KC1O4
removed
by
centrifuging,
and
free

purines
determined
on
the
supernatant.
A
shorter
pro
cedure
is
to
precipitate
the
acid
suspension
of
yeast
directly
with
Kj,HPO4
without
first
centrifuging
off
the
cells;
but
once
the
extracts

are
neutralized,
the
analysis
should
be
completed
the
same
day.
Results
DNA
estimation.—The
method
was
tested
on
four
separate
batches
of
pressed
yeast,
all
of
which
had
a
total
guanine

-|-
adenine
content
in
the
region
of
lOOjamol/g
dry
matter
(Table
II).
Between
analyses,
the
yeast
was
stored
at
4°C
(for
periods
of
up
to
3
weeks).
Free
guanine
in

the
DNA
fraction
amounted
to
1
-8—2-1
/imol/g
yeast
DM,
and
adenine,
3-O-3-5
/xmol/g.
For
12
estimations
on
batch
Y91115,
the
standard
deviation
was
±005
and
±007/xmol/grespectively.
The
mean
molar

ratio
adenine/guanine
was
1-73
±
0-02
(SE, 12
determinations)
corresponding
to
a
GC
content
of
37%,
in
agreement
with
published
data
for
yeast
DNA.10
Theoretically,
a
double-
stranded
DNA
of
this

base
composition
should
contain
1
mole
purine
in
618
g;
hence
the
percentage
by
weight
of
DNA
in
the
samples
of
yeast
examined
=
00618
x
^mol/g
(guaninc
+
adenine)

=
0-30-0-35
%,
as
found
previously
by
the
diphenylamine
procedure.10
No
difference
was
found
between
pressed
yeast
and
freeze-dried
preparations
(Table
I).
Total
purine
content
of
the
DNA
fraction,
as

determined
after
acid
hydrolysis,
was
the
same
as
the
free
purine
content
(Table
1).
Thus
digestion
with
alkali
was
effective
in
extracting
RNA
from
the
yeast
cells
and
rendering
it

acid-soluble.
If,
after
this
step,
the
suspension
was
not
acidified,
the
cells
being
simply
centrifuged
and
washed
with
water,
75
%
of
the
DNA
was
recovered
bound
to
the
cells

(Y90621,
Table
I).
Cold,
dilute
alkali
is
thus
a
poor
extractant
for
yeast
DNA,
pre
sumably
because
the
molecules
arc
too
large
to
pass
through
the
pores
of
the
cell

wall.4-10
Extraction
of
the
nucleotidc
pool
by
water
at
80°C
before
the
alkali
treatment
did
not
affect
the
DNA
content
(Y90727,
TABLE
I.
DNA
Purinc
Content
of
Commercial
Baker's
Yeast.

Yeast
batch
Y90621
Y90727
Y90823
Y9U15
Procedure
standard
alkali
digest
not
acidified
yeast
frcczc-dricd
standard
nuclcotidcs
first
extracted
with
water
at
80°
nuclcotidcs
first
extracted
with
0-5m
TCA
standard
nucleotidcs

first
extracted
with
PCA
standard
standard
nuclcotidcs
first
extracted
with
TCA
buffer
alkali
digest
acidified
with
acetic
acid
to
pH4-5
standard
yeast
residues
not
removed
before
pre
cipitation
of
KCIO4

Guaninc
urnol/g
Free
1-9
1-3
185
1-8
18
1-6
1-8
1-6
21
21
1-9
1-9
1-8
1-8
Total
1-8
1-3
21
1-9
2-3
Adeninc
ftmol/g
Free
3-3
2-5
3-2
31

305
2-8
3-2
2-7
3-4
3-5
3-2
35
30
31
Total
3-2
2-5
3-5
3-2
4-3
Table
I).
But
the
results
were
down
by
8-12%
when
PCA
or
TCA
was

used,
possibly
due
to
partial
depurination
at
low
pH.
It
was
thought
that
the
acid
precipitation
and
washing
steps
in
the
DNA
analysis
procedure
might
cause
a
similar
loss;
but

precipitation
of
the
alkali
digest
with
acetic
acid
at
pH
4-5,
followed
by
washing
with
0-1m
acetate
buffer
of
the
same
pH,
did not
significantly
affect
the
free
purine
figure,
though

total
purines
went
up
by
20%,
indicating
some
adsorption
on
the
cell
residue
of
RNA
breakdown
products.
The
nucleotidc
pool.—Commercial
baker's
yeast
was
found
to
contain
8-9
/xmol/g
more
adenine

than
guanine,
a
reflection
of
the
predominance
of
adenine-based
constituents
in
the
nucleotide
pool.0-11
(The
DNA
fraction
contributes
1
/imol/g
more
adenine
than
guanine,
but
this
is
offset
by
the

slight
excess
of
guanine
over adenine
in
yeast
RNA.18)
To
extract
nucleotides
rapidly
and
completely
from
yeast
is
difficult,5
especially
if
it
is
required
to retain
DNA
and
RNA
within
the
cell.8

This
is
due
to
the
presence
of
an
entire
cell
wall.
Many
reagents
close
up
the
cell
structure,
so
that
even
small
molecules
diffuse
out
slowly,
or
may
be
completely

trapped;
ethanol
is
an
example.10
The
most
commonly
used
extractants
are
the
strongly
acidic
protein
precipitants
perchloric
acid
(PCA)
and
trichloroacetic
acid
(TCA).
Their
anions
are
chaotropic,
i.e.,
they
destabilize

bio-membranes
and
enzyme
complexes.3
As
it
is
largely
undissociated,
TCA
penetrates
the
yeast
cell
more
rapidly.
At
23°C,
0-25m
TCA
extracted
0-8
jttmol/g
of
total
guanine
from
the
washed
cells

of
yeast
Y91004
after
0-25
h.
The
amount
extracted
increased
slowly
with time
to
1-1
/xmol/g
after
0-75
h,
then
more
rapidly,
to
3-1
/xmol/g
after
2
h.
The
amount
of

adenine
extracted
also
increased,
in
such
a
way
that
the
difference
(adenine-guanine)
remained
constant;
for
13
observations
made
with
different
concentrations
of
acid
the
mean
difference
was
9-6/xmoI/g
(SD
±

0-3).
This
suggests
that
the
increase
in
guanine
and
adeninc
with
increasing
time
was
due
to
the
extraction
of
RNA,
in
which
the
two
purines
are
present
in
nearly
equal

amount.
Extraction
with
0-25m
TCA
for
0-25-O-5
h
at
room
tem
perature
was
considered
satisfactory
for
removing
the
nucleo
tidc
pool
from
yeast
preparatory
to
analysis
for
nucleic
acids.
Vol.

86,
1980]
trevelyan:
determination of
dna
in
Saccharomyces
cerevisiae
TABLE
II.
Content
of
Total,
Nucleic
Acid,
and
Nucleolide
Purinc
in
Baker's
Yeast.
173
Yeast
batch
Y90621
Y9062I"
Y90727
Y90823
Y91115
Nucleolidc

pool
extraction
TCAB
PCA
W
TCA
TCAB
PCA
Total
;imolj
Guaninc
46-9
470
45-1
45-6
45-2
44-9
48-2
purine
'g
DM
Adenine
55-2
550
521
539
53-5
54-1
56-4
RNA

-I-
jim
Guaninc
45-3
44-5
440
44-3
47.7
b
48-1
e
DNA
purine
ol/g
DM
Adenine
44-3
43-8
43-2
43-6
47.31.
47
-6C
Nucleotide
(*mol/g
Guaninc
0-8
1-4
10
0-8

0-8
purinc
DM
Adenine
9-7
9-6
11-4
8-7
8-8
PCA,
perchloric
acid.
TCA,
chilled
0-5m
trichloroacetic
acid.
TCAB,
buffered
TCA.
W,
water
at
80"C.
a
Freeze-dried.
b
Standard
procedure.
c

Sequential
extraction
procedure.
Extraction
was
incomplete
after
0-5
h
with
a
weaker
reagent
(01
25m).
A
solution
containing
O-25m
sodium
trichloroacetate
in
addition
to
025m
TCA
was
preferred,
as
giving

better
buffering
power;
the
pH
of
the
extract
was
11,
close
to
the
value
at
which
yeast
RNA
is
least
soluble21
(Methods, pro
cedure
(B)).
Perchloric
acid
is
in
many
ways

a
more
convenient
reagent
than
TCA.8
A
0-2m
solution,
which
established
a
pH
of
10,
was
satisfactory
with
air-dried
yeast,1"
in
which
the
cell
membrane
has
been
damaged,
but
was

ineffective
with
pressed
yeast,
extracting
only
1
/xmol
of
adenine/g
DM
after
0-5
h.
A
concentration
of
1m
PCA
was
required
for
rapid
destruction
of
the
permeability
barrier
in
the

whole
population of
yeast
cells;
by
diluting
the
suspension
to
reduce
PCA
concentration
of
0-2m
after
5
min,
extraction
of
RNA
was
minimized
(Methods,
procedure
(D)).
Otherwise
it
reached
one-third
of

the
total after
0-5
h.
Another approach
was
to
add
a
lipo-
philic
reagent
(acetic
acid;
Methods,
procedure
(Q)
as
well
as
0-2m
PCA.
Nucleotides
diffused
from
the
yeast
cells
when
a

suspension
in
water
was
heated
rapidly to
80°C
{Methods,
procedure
(A)).
The
excess
of
nucleotide
adenine
over
guaninc,
as
found
by
this
method,
was
1-2/umol/g
greater
than
when
yeast
was
extracted

with
TCA
or
PCA
(Table
II).
These
methods
must
be
regarded
as
giving
only
an
approxi
mate
estimate
of
nucleotide
adenine,
and
especially
guanine,
but
are
suitable
for
extracting
the

nucleotide
pool
from
yeast
as
a
preliminary
to
analysis
of
nucleic
acid.
The
choice
between
them
will
depend
on
considerations
such
as
the
high
UV
absorption
of
TCA,
or
the

interference
of
PCA
with
Kjeldahl
digestion.
DNA
+
RNA
guanine
and
adenine.—Determinations
had
a
standard
deviation
of
±0-5-1
jitnol/g.
By
subtracting
the
purines
contributed
by
DNA,
the
content
of
RNA

guanine
and
adenine
could
be
found;
for
batch
Y90727
(Table
11)
this
was
42-5
jumol
guanine
and
40-6
/nmol
adenine/g
DM,
a
molar
ratio
of
1-05
as
compared
with
1-08

found
for
a
commercial
preparation
of
yeast
RNA."
Discussion
The
development
of
the
method
for
DNA
described
here
now
enables
the
three
broad
nucleic
acid
fractions
usually
distinguished
in
the

routine
analysis
of
microbial
cells4
to
be
determined
by
reference
to
a
single
well-defined
standard,
a
solution
of
adenine
and
guanine
in
1m
perchloric
acid,
which
is
stable
over
a

long
period
of
time.
The
separate
determination
of
guanine
and
adenine
has
proved
valuable
in
the
interpreta
tion
of
results.
We
have
found
the
simple
ion-exchange
procedure
for
estimating
these

purine
bases
to
be
convenient
and
reliable.
The
numerical
value
of
the
factor
0062
used
to
convert
DNA
guaninc
+
adenine
content
(as
jurnol/g
yeast) to
%
DNA
is
not
very

sensitive
to
the
exact
base
composition
assumed
for
(double-stranded)
DNA.
There
is
a
little
more
un
certainty
in
the
case
of
RNA.
Data
on
the
base
composition
of
5.
cerevisiae

RNA
given
by
Crestfield
et
at.,2
correspond
to
the
factor
0061,
as
do
the
slightly
different
results
of
Storck.15
Whatever
factor
is
chosen,
it
should
be
regarded
as
a
conven

tion
analogous
to
the
multiplier
6-25
used
to
convert
total
N
to
crude
protein.
As
nucleic
acids
contain
about
16%
N,
an
estimate of
the
protein
content
of
yeast
can
be

obtained
by
subtracting
the
percentage
of
nucleic
acid
from
a
crude
protein
figure
calcula
ted
as
total
N
in
pool-free
cells
x
6-25.
A
more
direct
method
is
to
extract

yeast
with
1m
PCA
(as
in
the
estimation
of
total
purine
content),
wash
the
cells
with
water,
disperse
them
in
water
+
a few drops
of
1
m
NaOH,
and
determine
nucleic

acid-
free
crude
protein
as
%
total
(Kjeldahl)
N
x
6-25
(about
40%
for
baker's
yeast).
PCA
at
room
temperature
extracts
little
protein
from
yeast;
in
the
extract
from
pool-free

yeast,
80%
of
solids
and
of
total
N
could
be
accounted
for
as
RNA.
References
1.
Carter,
B.
L.
A.,
Advances
in
Microbial
Physiology,
1978,
17,
243.
2.
Crestfield,
A.

M.,
Smith,
K.
C.
&
Allen,
F.
W.,
Journal
of
Biological
Chemistry,
1955,
216,
185.
3.
Hanstein,
W.
G.,
Davis,
K.
A.
&
Hatefi,
Y.,
Archives
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Biochemistry
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1971,
147,
534.
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Herbert,
D.,
Phipps,
P.
J.
&
Strange,
R.
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by
Norn's,
J.
R.
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D. W.,
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1971,
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Kopperschlager,
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H.
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1975,
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Sakaguchi,
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E.
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Suomalaincn,
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14,
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Pomcranz,
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122.
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Storck,
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B.
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Molecular
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New
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W.
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Trcvelyan,
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1975,
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Trevelyan,
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1977,
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Trevelyan,
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